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Presents an abstract of the article "Submicroscopic Duplication Detected During QF-PCR-based Trisomy Analysis; Characterisation of the Polymorphic D13S742 (13q12.12) Imbalance," by Kathy Mann, J. Worthington, A. F. Davies, and C. Mackie Ogilvie.

A multiplex of 12 primer pairs has been developed, allowing amplification of tetranucleotide STRs and copy number detection of chromosomes 13, 18 and 21 by QF-PCR. This assay has been used to test 4644 amniotic fluid samples, 1127 chorionic villi samples, 232 fetal tissue samples and 179 fetal and neonatal blood samples. 97.4% of the prenatal samples showed results consistent with the follow-up karyotype, including 321 trisomies and 10 triploids. Nine cases were trisomy mosaics. 103 cases could not be interpreted due to the presence of a second genotype (associated with blood staining and therefore likely to represent maternal cell contamination). In four samples, PCR failed to amplify the DNA. Since the development of the current multiplex, applied in the previous 12 months, two samples have been uninformative for a single chromosome. In five cases, QF-PCR indicated trisomy for one STR marker, while other markers on the same chromosome gave disomic results. In all these cases, the imbalance was found to be inherited and was not detected by karyotype analysis. These submicroscopic imbalances are considered likely to represent genomic duplication, either in tandem or at some distant locus. In the last calendar year, 96% of results were reported within one working day. The sensitivity of this test for the detection of non-mosaic trisomy is 331/331 = 1.00 (95% CI: 0.9910- 1.0000), and the specificity is 5290/5290 = 1.00 (95% CI: 0.9994- 1.0000). [ABSTRACT FROM AUTHOR]

Autosomal chromosome aneuploid pregnancies that survive to term, namely, trisomies 13, 18, and 21, account for 89% of chromosome abnormalities with a severe phenotype identified in prenatal samples. They are traditionally detected by full karyotype analysis of cultured cells. The average reporting time for a prenatal karyotype analysis is approximately 14 days, and in recent years, there has been increasing demand for more rapid prenatal results with respect to the common chromosome aneuploidies, to relieve maternal anxiety and facilitate options in pregnancy. The rapid tests that have been developed negate the requirement for cultured cells, instead directly testing cells from the amniotic fluid or chorionic villus sample, with the aim of generating results within 48 h of sample receipt. Interphase fluorescence in situ hybridization is the method of choice in some genetic laboratories, usually because the expertise and equipment are readily available. However, a quantitative fluorescence (QF)-PCR-based approach is now widely used and reported as a clinical diagnostic service in many studies. It may be used as a stand-alone test or as an adjunct test to full karyotype or array CGH analysis, which scan for other chromosome abnormalities not detected by the QF-PCR assay.

With advancing technology and the consequent shift towards an increasing application of molecular genetic techniques (e.g., microarrays, next-generation sequencing) with the potential for higher resolution in specific contexts, as well as the application of combined testing strategies for the diagnosis of chromosomal disorders, it is crucial that cytogenetic/cytogenomic services keep up to date with technology and have documents that provide guidance in this constantly evolving scenario. These new guidelines therefore aim to provide an updated, practical and easily available document that will enable genetic laboratories to operate within acceptable standards and to maintain a quality service.

Objective: To present the results of 10 years of quantitative fluorescence PCR (QF-PCR) analysis of prenatal samples for the rapid diagnosis of the common aneuploidies. This represents the largest QF-PCR data set from a single testing centre., Methods: QF-PCR analysis using a single assay containing 17 microsatellite markers was applied to all prenatal samples for the identification of trisomies 13, 18 and 21 and triploidy. A separate assay containing 14 sex chromosome markers was targeted to prenatal samples at increased risk of monosomy X., Results: Results from 40,624 prenatal samples comprising 14,144 chorionic villus and 26,480 amniotic fluid samples are summarised. A QF-PCR result was not possible for 2.24% amniotic fluid and 0.25% chorionic villus samples because of the presence of an additional genotype consistent with maternal cell contamination. Just 0.08% samples were uninformative for one or more chromosomes and 0.05% of samples failed to produce a genotype. Ninety-eight percent of samples were reported the following working day from sample receipt. Consumable costs were £ 5/sample., Conclusion: QF-PCR analysis is proven to be an accurate, robust and efficient method for the rapid diagnosis of common aneuploidies in prenatal samples. It has the advantage of detecting triploidy and mosaicism and benefits from considerable economy of scale., (© 2012 Crown copyright.)

QF-PCR refers to the amplification of chromosome-specific polymorphic microsatellite markers using fluorescence-labelled primers, followed by semi-quantitative analysis of the products on a genetic analyser to determine copy number and/or imbalances of specific chromosomal material. This approach is now widely used for rapid prenatal diagnosis of the common trisomies. In addition, it can successfully detect maternal cell contamination and mosaicism in prenatal material.